CELL BY DATE UPDATE29.08.0714:00

Presentation Outline

CBD Background Project Approach What’s Done

Lab Team Modelling Team Protocols Team

What’s Next Q & A

Background

Specifications Reports Thermal Exposure Accurately predict level of meat

spoilage Use of cell-free systems

Background Project Approach What’s Done What’s Next Q & A

CBD Reporter System Effects of temperature on gene

expression Constitutive promoter Fluorescent protein

(Example of a CBD DNA Construct)

Project Approach

Phase 1

Aim: Initial Testing Build DNA

constructs Initial Testing in

vivo Initial Testing in

vitro Generate

equations for Modelling

Phase 2

Aim: Characterization of Specific Construct

Operating Range Effects of Temp.

on Fluorescence vs.

Time Lifespan

Generate preliminary data for Modelling

Phase 3

Aim: Testing/Validation of Predictive Model

Accurate prediction of outcome

Fluorescence vs. Time

Lifespan Corroborate specs

of device DsRed-Express &

in-veso

Background Project Approach What’s Done What’s Next Q & A

Project Approach: Breakdown

Background Design Phases What’s Done What’s Next Q & A

Lab Team Modelling Team Protocols Team

Build / amplifyDNA constructs Derive equations /

Carry out simulations

Gather / mergeprotocols

Conduct Expts /Generate data for

analysisGeneration of predictive model

What’s Done

Lab Team Cloning/Amplification

pTet-GFP pT7-GFP

Biobricks construct does not work well

To be obtained from different sources

pCI-GFP (Cancelled) Awaiting DsRed-

Express

Background Project Approach What’s Done What’s Next Q & A

Modelling Team

kFP : Function of Temperature. k is based on the promoter used as promoters take time to turn on.

dFP : Function of System. May be considered to be a function of temperature as proteins degrade faster at higher temperatures.

What’s Done

Background Project Approach What’s Done What’s Next Q & A

Modelling Team

What’s Done

Background Project Approach What’s Done What’s Next Q & A

What’s Done

Protocols Team

Background Project Approach What’s Done What’s Next Q & A

Works in vivo & in vitro Tested @ 10oC, 25oC, 37oC,

45oC Does not work at 10oC, 45oC But when 10oC left o/n at

25oC, gene expression occurs

Worked weakly in vivo & in vitro

Very weak output cf. pTet To be obtained from

different sources and tested

What’s Done Protocols Team

pTet in Vitro - 10 Degrees

0

5000

10000

15000

20000

25000

30000

00:00 01:00 02:00 03:00 04:00 22:00 05:30

Sample

Fluo

resc

ence

A1 - pTet in Vitro

A1 - pTet in Vitro

A1 - pTet in Vitro

GFP dilution

Cell extract only

Blank control

Blank control

Blank control

Background Project Approach What’s Done What’s Next Q & A

pTet Vitro - 37 degrees

0

5000

10000

15000

20000

25000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Hours (aprx)

Flu

ore

sc

en

ce

pTet 1

pTet 2

GFP

Neg. Control

(Gaps in data due to lab restrictions)(1 magnitude increase)

What’s Done Protocols Team

pT7 Vitro 37oC

0

5000

10000

15000

20000

25000

30000

00:00 00:30 01:00 01:30 02:00 02:30 03:00 03:30 04:00 22:00 02:30 05:30

Sample

Fluore

scen

ce

A1 - pT7 in Vitro

A2 - pT7 in Vitro

A3 - pT7 in Vitro

Cell extract only

GFP dilution

Blank control

Blank control

Blank control

Background Project Approach What’s Done What’s Next Q & A

Fluorescence Vs Time

0

20000

40000

60000

80000

100000

120000

140000

0.000 20.000 40.000 60.000 80.000 100.000

Time (sec)

Fluo

resc

ence

A1 pTet

A2 pTet

A3 pTet

A5 LB-Media

A6 LB-Media

A7 LB-Media

B1 pT7-GFP

B2 pT7-GFP

B3 pT7-GFP

B4 LB-Media + Non expressive culture

B5 LB-Media + Non expressive culture

B6 LB-Media + Non expressive culture

C1 Diluted GFP

C2 Diluted GFP

C3 Diluted GFP

H10 Blank Control

H11 Blank Control

H12 Blank Control

(Not working well)(Difference in expression in

vivo)

What’s Done

Background Project Approach What’s Done What’s Next Q & A

Phase 1

Aim: Initial Testing Build DNA

constructs Initial Testing in

vivo Initial Testing in

vitro Generate

Equations for Modelling

Phase 2

Aim: Characterization of Specific Construct

Operating Range Effects of Temp.

on Fluorescence vs.

Time Lifespan

Generate preliminary data for Modelling

Phase 3

Aim: Testing/Validation of Predictive Model

Accurate prediction of outcome

Fluorescence vs. Time

Lifespan Corroborate specs

of device DsRed-Express &

in-veso

What’s Next

Timeline

Background Project Approach What’s Done What’s Next Q & A

Week 8

Week 9

Week 10

Phase 2 [GFP] & δ[GFP] curves Homemade cell

extract

Phase 2 Effects of temperature Gentle / steep gradients Preliminary data analysis

Phase 3 Suitable temp. step

increases Refine predictive

model

Phase 4? Proper documentation Substitute GFP w/ DsRed-Express In-veso gene expression Packaging Methods

Q & A

Background Project Approach What’s Done What’s Next Q & A